Inhaltsangabe
Until the mid 1980s, the detection and quantification of a specific mRNA was a difficult task, usually only undertaken by a skilled molecular biologist. With the advent of PCR, it became possible to amplify specific mRNA, after first converting the mRNA to cDNA via reverse transcriptase. The arrival of this technique―termed reverse transcription-PCR (RT-PCR)―meant that mRNA suddenly became amenable to rapid and sensitive analysis, without the need for advanced training in molecular biology. This new accessibility of mRNA, which has been facilitated by the rapid accumulation of sequence data for human mRNAs, means that every biomedical researcher can now include measurement of specific mRNA expression as a routine component of his/her research plans. In view of the ubiquity of the use of standard RT-PCR, the main objective of RT-PCR Protocols is essentially to provide novel, useful applications of RT-PCR. These include some useful adaptations and applications that could be relevant to the wider research community who are already familiar with the basic RT-PCR protocol. For example, a variety of different adaptations are described that have been employed to obtain quantitative data from RT-PCR. Quantitative RT-PCR provides the ability to accurately measure changes/imb- ances in specific mRNA expression between normal and diseased tissues.
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RT-PCR, the most widely used technique in biomedical research for analyzing gene expression, has proven a significant boon to the molecular investigation of disease pathogenesis. In RT-PCR Protocols, Joe O'Connell has assembled a panel of highly regarded molecular biologists and clinical researchers to describe in detail their most novel, useful, and interesting RT-PCR applications. Here the newcomer will find readily reproducible protocols for highly sensitive detection and quantification of gene expression, the in situ localization of gene expression in tissue, and the cloning of genes, as well as for analyzing T-cell clones and the differential expression of genes. For the expert seeking to extend the usefulness of RT-PCR, there are user-friendly applications that complement the latest technological advances, including laser-capture microdissection (LCM), real-time and quantitative PCR, microarray technology, cDNA cloning, and antibody engineering. Each protocol emphasizes the technical steps critical for experimental success and includes tips on avoiding potential pitfalls, notes explaining the method's scientific basis and its possible sources of error, and comments on how it might best be accommodated to various experimental circumstances.
Diverse and wide-ranging, RT-PCR Protocols offers investigators powerful techniques to be used in the molecular investigation of disease pathogenesis and shows how RT-PCR can complement other technological advances in the design of today's new therapeutic strategies.
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